Gene therapy and antisense treatment methods are currently used practically for treating congenital genetic diseases, cancer cells or AIDS by introducing an intended gene, antisense oligonucleic acid or derivative thereof into cells and expressing that gene or function, and studies are being conducted on various types of vectors for use as carriers for introducing genes (DNA), antisense oligonucleic acids and derivatives thereof into cells.
Research is being conducted on cationic substances such as cationic polymers, cationic liposomes and cationic lipids for use as one such type of vector in the form of a non-viral vector that eliminates concerns over safety, has favorable efficiency, is free of immunogenicity and is easily prepared.
In methods using these cationic substances, since complexes of DNA and cationic substances are positively charged, aggregation ends up occurring due to interaction with blood cells and blood components such as albumin, thereby impairing the delivery to the target cells. Studies have been conducted on various methods to solve this problem, including coating a complex of nucleic acid and cationic polymer with a hyaluronic acid derivative (Patent Document 1: Japanese Unexamined Patent Publication No. 2005-176830); coating with polyethylene glycol (PEG) having a carboxyl group-containing side chain and sugar residue-containing side chain (Patent Document 2: Japanese Unexamined Patent Publication No. 2003-231748); and preventing complex aggregation by using PEG having a free carboxylic acid pendant group (Non-Patent Document 1: J. Biomater. Sci. Polymer Edn., Vol. 14, No. 6, pp. 515-531 (2003)).
Complexes of DNA and cationic substances modified in this manner exhibit low aggregation and exhibit favorable gene expression in cells. However, since these complexes are heterogeneous suspensions, they have poor storage performance, are required to be used promptly following preparation, and end up aggregating when prepared at high concentrations, thereby having the disadvantages of difficulty in adjusting concentration and bothersome handling. In addition, it was difficult to prepare these complexes with satisfactory reproducibility.
On the other hand, in the case of using the modified complex of DNA and cationic substance as described above to introduce a gene and the like, it is also important to control the size (particle diameter) of the complex. This is because, in the case of administering into blood or tissue, subsequent diffusion of the complex and the efficiency at which it is delivered and incorporated by cells greatly affect its pharmacological efficacy. In general, however, in the case of mixing ionic polymers such as cationic polymers to form a complex, the polymer ends up aggregating resulting in an increased likelihood of the formation of extremely large particles or fibrous complexes. In order to prevent this, it is necessary to make the concentrations of the solutions mixed extremely dilute. However, since preparations used for gene introduction and the like are required to have a certain minimal concentration, there was the problem of being unable to avoid the formation of large mass of aggregates. In addition, although methods are also considered consisting of first forming small complexes by mixing dilute solutions followed by concentrating, suitable means for concentration was unable to be achieved since the complex particles ended up rapidly aggregating.
Therefore, in order to solve these problems, studies were conducted on freeze-drying methods typically used to facilitate transport of biological preparations and enhance storage stability. However, since freeze-drying complexes of nucleic acids, oligonucleic acids or derivatives thereof with polycationic substances ends up impairment of the structure of the complex due to the freeze-drying, when used for gene introduction or introduction of oligonucleic acids, the complexes were confirmed to hardly demonstrate any functions as genes or antisense oligonucleic acids (Non-Patent Document 2: J. Pharm. Sci., Vol. 90, pp. 1445-1455 (2001)).
A method to solve these problems was proposed in which freeze-drying is carried out after adding a high concentration of monosaccharide or disaccharide (Non-Patent Document 3: Biochim. Biophys. Acta., 2000 Sep. 29, 1468 (1-2): 127-138). However, the amount of sugar required is 500 to 1000 times the amount of DNA in terms of the weight ratio, making this method impractical in consideration of the solution following rehydration having a much higher osmotic pressure than physiological conditions. In addition, monosaccharides and disaccharides do not offer advantageous effects for gene expression. In addition, the use of a neutral polysaccharide, dextran has been attempted to reduce osmotic pressure after rehydration (Non-Patent Document 4: J. Pharm. Sci., Vol. 94, pp. 1226-1236 (2005)). However, high molecular weight dextran greatly inhibits gene expression, and in the case of using low molecular weight dextran (molecular weight of about 3000), it was necessary to add dextran at a considerably high concentration of 100 times or more that of DNA in terms of the weigh ratio in order to prevent aggregation caused by freeze-drying (Non-Patent Document 4: J. Pharm. Sci., Vol. 94, pp. 1226-1236 (2005)). For use of this type of freeze-dried product in vivo, the freeze-dried product is required to be rehydrated with a small amount of water or solvent after freeze-drying to obtain the required concentration of DNA and then concentrated to a high concentration. As a result, the concentration of dextran following rehydration exceeds 10%, and there are limitations during the freeze-drying procedure such as on DNA concentration and cooling temperature, thereby making practical application difficult.    Patent Document 1: Japanese Unexamined Patent Publication No. 2005-176830    Patent Document 2: Japanese Unexamined Patent Publication No. 2003-231748    Non-Patent Document 1: J. Biomater. Sci. Polymer Edn., Vol. 14, No. 6, pp. 515-531 (2003)    Non-Patent Document 2: J. Pharm. Sci., Vol. 90, pp. 1445-1.455 (2001)    Non-Patent Document 3: Biochim. Biophys. Acta., 2000 Sep. 29, 1468 (1-2): 127-138    Non-Patent Document 4: J. Pharm. Sci., Vol. 94, pp. 1226-1236 (2005)